The broad objective of this proposal is a better understanding of how metal-containing enzymes work. The more specific aims involve characterization of two classes of Fe-based metalloenzymes with 'unusual'active sites - nitrogenase and hydrogenase. The questions that we hope to answer revolve around molecular structure (what atoms are where) dynamics (how the atoms move), and electronic structure (how are electrons distributed among the various atoms). To achieve this knowledge, we have arranged a close collaboration between microbiologists, biochemists, and spectroscopists, including a long-term joint effort between the Harwood, Newton, and Cramer groups, as well as major contributions from many other labs. In the course of this project we will develop or enhance a variety of spectroscopic 'probes'that allow us to answer questions about the structure of enzyme intermediates. These include numerous x-ray and nuclear techniques based on modern synchrotron radiation sources - (XAFS, NRVS, SRPACS, and NFS). We will complement these with a novel laser technique - femtosecond pump-probe spectroscopy (FPPS). Finally, we will balance these 'large facility'methods with campus-based spectroscopies including far-infrared absorption, vibrational circular dichroism, and resonance Raman. For nitrogenase and hydrogenase, the questions that we plan to address are: How does structure change during the course of the catalytic cycle? Where do substrates and inhibitors bind? What are the undefined light atoms? Our spectroscopic techniques will allow us to monitor the enzyme active sites with emphasis on certain regions. We will use this capability to focus on structural and dynamic issues that are beyond the reach of protein crystallography. How is research on bacterial enzymes related to public health? Quite simply, nitrogenase and biological nitrogen fixation are essential components of the nitrogen cycle, a process responsible for more than half of the food we eat. For hydrogenase, a better understanding of its catalytic mechanism offers the promise of a future pollution-free 'hydrogen economy'. Apart from specific knowledge about these two enzymes, the techniques developed in the course of this study will be applicable to the hundreds of metalloproteins involved in human metabolism.